5,7,3',4'-tetramethoxyflavone (TMF), one of the major polymethoxyflavones (PMFs) isolated from Kaempferia parviflor, has been reported possessing various bioactivities including antifungal, antimalarial, antimycobacterial, and anti-inflammatory activities. Although several studies on the TMF have been reported, the information about the metabolism of TMF and the structures of TMF metabolites is still not yet clear. In this study, an isotope-labeling method was developed for the identification of TMF metabolites. Three isotope-labeled 5,7,3',4'-tetramethoxyflavones (5,7, 3',4'-tetramethoxy[3'-D3]flavone; 5,7, 3',4'-tetramethoxy[4'-D3]flavone; and 5,7, 3',4'-tetramethoxy[5,4'-D6]flavone ) were synthesized and administered to rat. The urine samples were collected and main metabolites were monitored by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry. Five TMF metabolites were unambiguously identified as 3'-hydroxy-5,7,4'-trimethoxyflavone, 7-hydroxy-5,3',4'-trimethoxyflavone sulfate, 7-hydroxy-5,3',4'-trimethoxyflavone, 4'-hydroxy-5,7,3'-trimethoxyflavone, and 5-hydroxy-7,3',4'-trimethoxyflavone.